Journal: Disease Models & Mechanisms

Article Title: Development of a physiologically relevant and easily scalable LUHMES cell-based model of G2019S LRRK2-driven Parkinson's disease

doi: 10.1242/dmm.048017

Figure Lengend Snippet: Phosphorylation of bona fide LRRK2 substrates in LUHMES clones. (A) Representative WB results showing the phosphorylation of two LRRK2 bona fide substrates, LRRK2 Ser1292 and RAB10 Thr73, in naïve and clonal LUHMES cells (L10WT and L14GS). LUHMES cells were differentiated for up to 4 days, and levels of total and phosphorylated LRRK2 and RAB10 were analyzed each day. GAPDH was used to ensure equal loading. The protein corresponding molecular mass (in kDa) is indicated on the left side of the panel. (B) Quantification of pRAB10 levels. Results from six independent experiments. Error bars show mean±s.d.

Article Snippet: Control proteins were recombinant full-length human his-tagged RAB10 (Novus Biological, catalog no. NBP2-23392) and human full-length LRRK2 (ThermoFisher Scientific, catalog no. 15383806).

Techniques: Phospho-proteomics, Clone Assay

Journal: Disease Models & Mechanisms

Article Title: Development of a physiologically relevant and easily scalable LUHMES cell-based model of G2019S LRRK2-driven Parkinson's disease

doi: 10.1242/dmm.048017

Figure Lengend Snippet: LRRK2 kinase inhibitor MLI-2 effectively reduces phosphorylation of two bona fide LRRK2 substrates. (A) LUHMES cells were differentiated for 2 days and then treated with increasing concentrations of LRRK2 kinase inhibitor MLI-2 for 4 h. A potent reduction in the phosphorylation of LRRK2 Ser1292 and RAB10 Thr73 was observed. (B,C) Dose-response curves of the LRRK2 kinase inhibitor MLI-2 against LRRK2 pSer1292 and RAB10 pThr73 in WT and G2019S LUHMES cells. The protein corresponding molecular mass (in kDa) is indicated on the left side of the panel. (B) Half-maximal inhibitory concentration (IC 50 ) values, calculated for WT and G2019S LRRK2 by GraphPad Prism software on LRRK2 pSer1292, were found to be 21.2 pM (L10WT clone, filled circles) and 1.45 nM (L14GS clone, filled squares). (C) IC 50 values, calculated by GraphPad Prism software for WT and G2019S LRRK2 on RAB10 pThr73, were found to be 0.78 nM (L10WT clone, filled circles) and 2.31 nM (L14GS clone, filled squares). Results from three independent experiments.

Article Snippet: Control proteins were recombinant full-length human his-tagged RAB10 (Novus Biological, catalog no. NBP2-23392) and human full-length LRRK2 (ThermoFisher Scientific, catalog no. 15383806).

Techniques: Phospho-proteomics, Concentration Assay, Software

Journal: bioRxiv

Article Title: Bone marrow-derived CCR5-expressing macrophages promote venous thrombus resolution by up-regulating plasminogen activators and VEGF

doi: 10.1101/2023.09.18.558360

Figure Lengend Snippet: (A) Ccr5 gene expression was determined by real-time RT-PCR. All values represent mean ± SEM (n = 5). (B) Immunohistochemical analysis was performed using anti-CCR5 in thrombus samples from WT mice. Representative results from 6 independent experiments are shown. (C) A double-color immunofluorescence analysis of CCR5-expressing cells in the thrombus at day 5. The samples were immunostained with the combination of anti-CCR5 and anti-F4/80. (D-F) The gene expression of Ccl3 (D), Ccl4 (E) and Ccl5 (F) were examined by real-time RT-PCR. All values represent mean ± SEM (n = 5). (G) Intrathrombotic contents of CCL5 in WT mice after IVC ligation. All values represent mean ± SEM (n = 4). (H) A double-color immunofluorescence analysis of CCL5-expressing cells in the thrombus at day 5. The samples were immunostained with the combination of anti-CCL5 and anti-F4/80. (I-S) IVC ligation-induced deep vein thrombus in WT and Ccr5 -/- mice. (I) Macroscopic appearance of thrombi in WT and Ccr5 -/- mice 10 days after IVC ligation. Representative results from 6 independent animals are shown. (J) Thrombus mass of WT and Ccr5 -/- mice at the indicated time intervals after IVC ligation. All values represent the mean ± SEM (n = 5). ** P < 0.01; * P < 0.05, vs. WT. (K and L) Histopathologic analysis of thrombi obtained from WT and Ccr5 -/- mice. Thrombi were stained with HE (K) or Masson trichrome solution (L). (M) The measurement of IVC blood flow by laser Doppler imaging. All values represent the mean ± SEM (n = 5). ** P < 0.01, vs. WT. (N) Immunohistochemical analysis was performed using anti-CD31 on thrombus samples from WT and Ccr5 -/- mice. Representative results from 6 independent experiments are shown (day 14). Arrowheads indicate the CD31 + areas with tube-like formation. (O) CD31 + vascular areas were determined (n = 5). * P < 0.05, vs. WT. (P) Thrombus mass of vehicle- and Maraviroc-treated WT mice at day 6 after IVC ligation. Values represent the mean ± SEM (n = 5). * P < 0.05, vs. vehicle-treated controls. (Q) The measurement of IVC blood flow by laser Doppler imaging. All values represent the mean ± SEM (n = 5). * P < 0.05, vs. vehicle-treated controls. (R) Thrombus mass of PBS- and rCCL5-treated WT mice at day 6 after IVC ligation. Values represent the mean ± SEM (n = 5). * P < 0.05, vs. PBS-treated controls. (S) The measurement of IVC blood flow by laser Doppler imaging. All values represent the mean ± SEM (n = 5). * P <0.05, vs. PBS-treated controls.

Article Snippet: Recombinant mouse CCL3, CCL4 and CCL5 were purchased from R&D Systems (Minneapolis, MN).

Techniques: Gene Expression, Quantitative RT-PCR, Immunohistochemical staining, Immunofluorescence, Expressing, Ligation, Staining, Imaging

Journal:

Article Title: The voltage-gated potassium channel Kv1.3 is highly expressed on inflammatory infiltrates in multiple sclerosis brain

doi: 10.1073/pnas.0501770102

Figure Lengend Snippet: Expression of Kv1.3 on inflammatory cells in MS brain. Paraffin sections were stained by indirect immunoperoxidase for Kv1.3, CD3, CD4, CCR7, and CCR5. Areas used in sectioning were from a white matter plaque. There were many perivascular inflammatory cells that stained positively for CD3 (A), Kv1.3 (B), and CD4 (B Inset) on consecutive sections. (C) Kv1.3 was also localized on inflammatory cells in the white matter parenchyma (Inset reveals membrane polarization of Kv1.3 staining). (D) Consecutive sections through another perivascular infiltrate revealed numerous Kv1.3+ inflammatory cells, which were predominantly CCR7- (E) (Inset reveals rare CCR7 positive staining), and CCR5+ (F). (Scale bar, 50 μmin A and B and 20 μmin C, D, E, and F.)

Article Snippet: The CCR7 antibody was from R & D Systems.

Techniques: Expressing, Staining, Membrane

Journal:

Article Title: The voltage-gated potassium channel Kv1.3 is highly expressed on inflammatory infiltrates in multiple sclerosis brain

doi: 10.1073/pnas.0501770102

Figure Lengend Snippet: Confocal microscopy of Kv1.3 and CD3/CD4 in MS brain tissue. Confocal microscopic images of 20-μm floating sections of postmortem MS brain tissue. (A and B) Parenchymal infiltrate with positive CD3 and punctate Kv1.3 staining in the membrane. (C) A cluster of lymphocytes stained positively for Kv1.3 and CD4, as well as occasional CD8 (Inset). (D) CD3+ cells showed extensive Kv1.3 and CD3 colocalization in the membrane. Immunofluorescent staining of PB-derived T cells concentrated on a slide by cytospin showed membrane patterns of staining of CD4, CD8, and Kv1.3. (E) Day 7-activated naive and TCM are CD4+, CCR7+ (data not shown) and Kv1.3-.(F) Chronically stimulated resting TEM were CD4+, CCR7- (data not shown) and expressed Kv1.3 in the membrane but with minimal colocalization with CD4. (G)TEM that had been recently activated expressed increased amounts of Kv1.3 and demonstrated more colocalization with CD4 similar to the brain tissue lymphocyte in D. (H) Naive CD8+ T cells expressed no Kv1.3 but, when chronically stimulated and activated (I), exhibited intense Kv1.3 expression and colocalization with CD8. (J) Isotype controls using nonspecific rabbit primary antibody followed by usual secondary label and with primary rabbit anti-human and nonspecific labeled mouse anti-rabbit secondary antibody showed no background staining.

Article Snippet: The CCR7 antibody was from R & D Systems.

Techniques: Confocal Microscopy, Staining, Membrane, Derivative Assay, Expressing, Labeling

Journal:

Article Title: The voltage-gated potassium channel Kv1.3 is highly expressed on inflammatory infiltrates in multiple sclerosis brain

doi: 10.1073/pnas.0501770102

Figure Lengend Snippet: Immunofluorescence and electrophysiology of peripheral and CSF T cells. FACS plots revealed staining patterns of CCR7 vs. CD45RA. (A) CD4-gated MS-derived CSF cells revealed a predominantly TCM phenotype immediately ex vivo, but with rapid conversion to short-lived TEM after 7 and 14 days of stimulation using anti-CD3/CD28 coated beads (B and C). By comparison, negatively sorted CSF derived from noninflammatory neurological controls showed CD4+ cells that were predominantly TCM (D) and remained TCM at day 7 despite identical manner of stimulation (E). (F-H) CD4 cells derived from PB were predominantly TCM and naive phenotype (F), and maintained high levels of CCR7 even after 7 and 14 days of stimulation using anti-CD3/CD28 coated beads (G and H). (I) Single cell patch-clamp analysis of MS-derived CSF cells revealed high Kv1.3 channel numbers per cell; mean channel number per cell was 1,082 ± 489 (mean ± SEM, n = 28). Shown for comparison on the right are mean Kv1.3 channel numbers from activated PB T cells from three MS patients (n = 32), and from 10 healthy controls (n = 33). (J and K) Family of currents. The test potential was changed from -60 to 60 mV in 10-mV increments every 30 s (V1/2 = -28 mV). (L) The current in CSF cells exhibited the characteristic use-dependence of Kv1.3 when 200-ms pulses were applied every second to 40 mV. (M and N) Pharmacological blockade with the potent and selective Kv1.3 inhibitor ShK(L5) (14) confirmed that the current is carried by Kv1.3. (O) Representative images of CSF resting (top) or activated (bottom) T cells stained with anti-Kv1.3 and CCR7 Abs. (P) Staining intensities for Kv1.3 and CCR7 in resting and activated CSF T cells. (Q) Z-stack of confocal images through an activated CSF T cell.

Article Snippet: The CCR7 antibody was from R & D Systems.

Techniques: Immunofluorescence, Staining, Derivative Assay, Ex Vivo, Comparison, Patch Clamp